(6S,7S)-7-Amino-3-(methoxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

The actual concentration of the test substance was determined at the start and at the end of a 72 hours incubation period in filtered samples of the cultures exposed to the test substance and also in one blank containing 100.0 mg test substance per L test medium (nominal concentration), but no algae.

Vehicle:

no

Details on test solutions:

The test substance was dissolved in nutrient medium, consisting of sterile double distilled water and sterile concentrated nutrient medium.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

Selenastrum capricornutum ATCC (American Type Culture Collection) 22662, obtained directly from ATCC, Rockville, Maryland 20852, USA.
Deep frozen algae were thawed and cultivated as described by the supplier in 250 mL conical flasks (Erlenmeyer) each containing 100 ml medium for stock culture at a temperature of 23 ± 2 °C under permanent light with an intensity of at least 6000 lux. The culture was maintained under the same conditions. In about weekly intervals 1 mL of the stock culture was diluted 100-fold with nutrient medium for precultivation and incubation was continued. Test precultures were prepared by incubating a new stock culture for three days. At the start of the experiment the cell density was determined and adjusted to about 10^5 algae/mL by diluting with nutrient medium for precultivation.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None.

Hardness:

Water hardness corresponds to the standard test medium.

Test temperature:

22 +- 1 °C.

pH:

The pH of the test culture at the start of the incubation period was lower than that of the controls (6.9 versus 8.3). 72 hours thereafter the pH of the test substance culture was 7.1 and was now comparable to the pH of 7.6 of the control culture. The pH of the blank containing test substance, but no algae, was 7.0 at the start and at the end of the incubation period.

Dissolved oxygen:

n.a.

Salinity:

N.a.

Nominal and measured concentrations:

Nominal: 100 mg/L.
Actual: At the start of the incubation, the actual test substance concentration in the test substance culture was 100.0 % of the nominal concentration. After 72 hours of incubation, the actual test substance concentration was 93.0 % of the actual starting concentration in the test substance culture. In the blank without algae, the actual test substance concentration was 99.7 % of the actual starting concentration. This indicates that the test substance was neither markedly degraded nor taken up by the algae during the incubation time of 72 hours.

Nutrient medium was used for the negative controls.
One blank containing 100.0 mg test substance per L test medium (nominal test substance concentration), but no algae, was incubated under the same culture conditions for analysis of the test substance concentration.

Details on test conditions:

Sterile nutrient medium was used for the negative control cultures.
The test culture and the blank were set up in 250 ml conical flasks. The total culture volume was 100 ml in each case. The complete cultures each consisted of 10 ml inoculum (10^5 algae/ml) and 90 ml of the test substance stock solution or of the nutrient medium (control).
The blank contained 10 ml of nutrient medium (instead of the algal inoculum) and 90 ml of the test substance stock solution.
Incubated for three days in permanent light with an intensity of at least 6000 lux (wavelength 400-700 nm) while shaking.

Reference substance (positive control):

not required

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Growth inhibition: The test substance did not markedly inhibit the growth of the algae during the incubation period of 72 hours (0.3 or 0.2 % inhibition, based on the area under the growth curves or based on the average growth rates).

Results with reference substance (positive control):

n.a.

Reported statistics and error estimates:

n.a.

Validity of the test

The increase in cell density in the control cultures during the 72 hours incubation period was about 60-fold, i.e. more than 16-fold as recommended in the EC-guideline, corresponding to about 5.9 generations and showing optimal growth conditions for the algae.

The actual test substance concentrations at the end of the incubation period were higher than 80% of the actual starting concentration.

Validity criteria fulfilled:

yes

Conclusions:

Based on the area under the growth curves or the average growth rate:
NOEC(0-72h) is >=100 mg/L.
EbC5072h >100 mg/L.
ErC5072h >100 mg/L.

Executive summary:

A Selenastrum capricornutum growth inhibition test according to the directive 92/69/EEC Part C.3 was performed to determine the possible effects of 7-AMCA on the growth of a unicellular green algal species.

A limit-test with one concentration of 100 mg of 7-AMCA per L test medium was performed. There were three replicates for each test and control culture. In each vessel the cell density of the algae was determined 24, 48 and 72 hours after the onset of incubation and the pH was measured at the onset and at the end of the incubation period. Possible test substance effects were determined by comparison of the areas under the growth curves and by comparison of the growth rates. The actual concentration of the test substance was determined at the start and at the end of a 72 hours incubation period in filtered samples of the cultures exposed to the test substance and also in one blank containing 100.0 mg test substance per L test medium (nominal concentration), but no algae.

Results

At the start of the incubation, the actual test substance concentration in the test substance culture was 100.0% of the nominal concentration. After 72 hours of incubation, the actual test substance concentration was 93.0 % of the actual starting concentration in the test substance culture. In the blank without algae, the actual test substance concentration was 99.7 % of the actual starting concentration. This indicates that the test substance was neither markedly degraded nor taken up by the algae during the incubation time of 72 hours. The pH of the test culture at the start of the incubation period was lower than that of the controls (6.9 versus 8.3). 72 hours thereafter the pH of the test substance culture was 7.1 and was now comparable to the pH of 7.6 of the control culture. The pH of the blank containing test substance, but no algae, was 7.0 at the start and at the end of the incubation period.

Increase in cell densities

During the 72 hours incubation period the cell density in the control culture was increased by a factor of about 60 (about 5.9 generations).

Growth inhibition

The test substance did not markedly inhibit the growth of the algae during the incubation period of 72 hours (0.3 or 0.2 % inhibition, based on the area under the growth curves or based on the average growth rates).

NOEC and EC50-values

Two "no observed effect concentrations" (NOECs) and two EC50 values were derived - based on the area under the growth curves and based on the average growth rates. As the actual test substance concentrations only minimally differed from the nominal ones and as they were stable throughout the incubation period the nominal starting concentrations are given: NOEC (0-72h) of 7-AMCA based on the area under the growth curves or the average growth rate is >=100 mg/L. EbC5072h >100 mg/L. ErC5072h >100 mg/L.

Description of key information

Based on the area under the growth curves or the average growth rate:  
NOEC(0-72h) is >=100 mg/L. 
EbC5072h >100 mg/L. 
ErC5072h >100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The EC50 is not 100 mg/L, as one is forced to enter in the field above, but the EC50 is >100 mg/L.